Plant tissue culture involves growing all or part of a plant from a culture of parent cells. A number of techniques exist for plant tissue culture and include two general types: solid based cultures, in which cells are grown in a gel or membrane, and liquid cultures, in which the cells are free to float in the medium, in a manner similar to fermentation. In both cases, the objective is to make air, vitamins, minerals and hormones accessible to the growing cells or plants. The technique of preference depends on the specific plant, and the stage at which it is growing.
Membrane rafts, comprising hydrophilic microporous membranes sealed to a plastic base, have been described by prior art workers. The raft forms a floating platform on which to seed the cells for plant tissue growth. It comprises a frame to whose bottom perimeter a microporous membrane is sealed. The membrane serves as the bottom of the raft. Liquid and nutrients are meant to reach the cells growing on the membrane only through the pores. The frame prevents liquid from washing over the sides of the membrane. The design of the raft is meant to fit the growing vessel containing the liquid medium. Both square and round designs are known.
Medium contained in the liquid below the membrane reaches the cells by a combination of diffusion and bulk flow through the membrane pores and provides the cells with nutrients for growth. With some plants, substantial increases in the rate of plant growth can be achieved in comparison to growth on agar gels or other solid plant cell support systems. Reports of these results can be formed in the published results of Hamilton RM, Pederson H and Chin C-K, "Biotechniques", vol 3, page 96 (1985) and Young R. E., Hale A., Camper N. D., Keese R. J., and Adelberg J. W., "Transactions of the American Society for Agricultural Engineers", vol 34, pages 328-333 (1991).
Although diffusion to and from a growing cell is quite slow in a gel medium, it can be one or two orders of magnitude faster in a liquid. In the case of the membrane raft, the cell is supported on a solid surface, but the nutrients are transported via a liquid through the pores. The larger the pore size and the greater the number of pores in the membrane, the faster the rate of transport to and from the cells. Membrane rafts therefore have the potential advantage of combining the best features of liquid and solid culture techniques. However, the structure of the prior art rafts is far from optimal.
The current commercial raft design is literally that of a simple flat bottomed "raft", with four legs (Sigma Chemical Co., St. Louis, Mo.). The raft floats as a result of the volume of water displaced, and like any raft, depends on the impermeability of its base to keep liquid out so that it remains afloat. For this reason, the membrane used for the base is restricted to a film with very small pores, and a low hydraulic permeability. Even then, this must be coupled with a lightweight design for the remainder of the raft.
The weight of the raft provides a downwards pressure on the membrane through which water flows. If the flow rate is greater than the ability of the growing cells to use such medium, the cells will die or grow very poorly. In order to be successful, the prior art structure requires a very specific balance between the raft displacement weight and the membrane properties.
The reason for increased rates of growth are not clearly known. However, one may hypothesize that they are related to rates of transport of nutrients to the cells, and cell generated inhibitors or waste products away from the cells. The faster the rate of transport, the higher the effective concentration of growth nutrients, and the lower the concentration of species which inhibit the growth.
Rates of transport will be faster the larger the pores and the greater the surface area thereof. Conventional membranes and porous materials cannot be used in the conventional prior art structure because their hydraulic permeability is too high. Under the weight of the raft over a 24 hour period, conventional rafts either accumulate too much liquid on the surface of the raft or sink completely.
It has been proposed to construct a raft frame with hollow walls or walls made from a foamed material. These solutions reduce the surface area available for cell growth while reducing the depth of displacement of the raft and thus do not provide an acceptable solution.
As the plants grow, the weight of the raft increases with time. This increases the depth at which the raft is floating, as well as the pressure causing liquid to move through the membrane. Although this may sometimes be a desirable occurrence, the prior art structure does not enable control over when and to what extent the two results occur.
A second problem with the prior art raft structure relates to the later stages of plant growth and, more particularly to the development of the plant's root system. At the point at which the culture medium has been changed to cause expression of both the root system and the leaves, the ideal is for the roots to be continuously immersed in the culture medium, with the leaves above the liquid surface. At this stage it is also important for the plant to have mechanical support from the root system to physically support the upper portion of the plant.
Since rootlets cannot penetrate the pores of the membrane, they have nothing in which to anchor themselves to provide this support. The above-mentioned Young et al reference describes two frame membrane and netting devices, or sandwiches of membrane and netting which attempt to solve this problem. These comprise netting, with a regular array of openings large enough for the rootlets to grow through and anchor, placed on top of a microporous membrane. However, this requires peeling away the upper layer of growing plants, which are embedded in the netting, from the bottom layers. This approach is clumsy, expensive, and harmful to the plants as some shock is caused to the root system when the plants are separated.
The patent literature includes various examples of floating apparatus suitable for use in plane cell tissue culture.
U.S. Pat. NO. 4,037,360 describes raft apparatus for growing plants by means of water culture including a buoyant body arranged to float in a stable position on a nutrient solution. The buoyant member contains a generally vertically oriented channel which accommodates the step of a plant whose root system extends into the nutrient solution below the member. To facilitate seed germination, the channel may include a porous or absorptive partition which provides fluid communication by capillary action with a location on which a seed may germinate. During plant growth, buoyancy of the body is increased by adding auxiliary buoyancy apparatus.
U.S. Pat. No. 4,382,348 describes a soilless plant growing device which includes a fine mesh plate supported only at its periphery by a buoyant frame. The buoyancy of the apparatus is such that seeds are maintained at the surface of and in contact with the nutrient solution during germination, while the fine mesh allows roots to subsequently grow into the solution.
U.S. Pat. No. 4,513,533 describes a method of growing plants in an open trough on a sheet of buoyant hydrophobic material such as polystyrene in which are formed a regular pattern of large holes. The holes are covered by a connected grid of plant collars, which hold seedlings or a solid support medium in which a seed has germinated. The buoyancy of the sheet is such as to maintain the plants above the surface of the water in the trough throughout the growth cycle.
U.S. Pat. No. 4,607,454 describes a floating bed for hydroponically germinating seeds. The floating bed is floatable and is hydrophobic.
U.S. Pat. No. 4,531,324 describes a plant tissue culture device in which the cultures are maintained in culture wells on a porous wick which dips into a liquid medium and transports nutrients and water to the cultures.
U.K. Patent 2,014,836 describes a method for producing a stand of plants in which a buoyant inert base is covered with an absorbent material and floated on a trough of liquid. The ends of the absorbent material extend beyond the base and dip into the liquid, thereby transporting nutrients and water to seeds on the surface of the absorbent material.